Streptococci diagnostic method

ABSTRACT

A METHOD OF DETECTING STREPTOCOCCAL ORGANISMS, ESPECIALLY GROUP A, B-HEMOLYTIC STREPTOCOCCI, IS PROVIDED WHEREIN SEPARATE SMEARS ON MICROSCOPE SLIDES COMPRISING NEGATIVE CONTROL AND POSITIVE CONTROL ORGANISMS ARE TREATED WITH ANTIBODY CONJUGATED TO PEROXIDASE, AND THE SLIDES THEN COMPARED UNDER THE MICROSCOPE, AFTER DYEING WITH ENZYME SUBSTRATES TO DETERMINE THE PRESENCE OF GROUP A STREPTOCOCCI.

"United States Patent O 3,790,447 STREPTOCOCCI DIAGNOSTIC METHOD ArthurAtsunobu Hirata, Waukegan, and William Franklin Boley, Lake Villa, Ill,assignors to Abbott Laboratories, North Chicago, Ill. N Drawing. FiledJuly 5, 1972, Ser. No. 268,967 Int. Cl. C12k 1/04 U.S. Cl. 195-103.5 R 3Claims ABSTRACT OF THE DISCLOSURE A method of detecting Streptococcalorganisms, especially Group A, ,B-hemolytic Streptococci, is providedwherein separate smears on microscope slides comprising negative controland positive control organisms are treated with antibody conjugated toperoxidase, and the slides then compared under the microscope, afterdyeing with enzyme substrates to determine the presence of Group AStreptococci.

DISCLOSURE OF THE INVENTION This invention relates to a new and novelmethod of utilizing labeled antibodies especially to detect the presenceof Group A Streptococci in, for example, a swab sample.

Serological grouping of human p-hemolytic Streptococcal infections isimportant to the medical profession, especially the presence of Group AStreptococci which necessitates therapy. As has been reported, asignificant error may result from the assumption that all fi-hemolyticStreptococci from the human nasopharynx belong to Group A.

Accurate and rapid identification of Group A 8-hemolytic Streptococcusas the causative agent in nasopharyngitis is a vital step towardsadequate treatment and eventual preveniont of the possible sequaelae ofthis infection, i.e., scarlet fever, rheumatic fever,glomerulonephritis. Such an identification of Group A Streptococcalpharyngitis requires laboratory assistance to demonstrate the organismdirectly.

A number of known methods are used to identify Group A Streptococcus.One involves resort to a fluorescent antibody that make for a specificidentification of infectious organisms. However, this laboratoryprocedure involves a costly fluorescent microscope. In another methodused in detecting Streptococci, a bacterial culture is grown. However,positive identification of Group A Streptococci requires 48 hours,involving in some cases an undesirable time lag of proper diagnosis andtreatment.

The method of the invention here incorporates advantages of sensitivity,rapidity, and use only of an ordinary light microscopy foridentification purposes rather than resorting to an expensivefluorescent microscope.

The diagnostic method here, in brief, involves a method of detecting thepresence of Group A Streptococci in the presence of other Streptococciand Staphylococci organisms in an unknown sample. First is prepared aseries of at least 3 dry slide smears, one of said smears acting as anegative control comprising a suspension of only Group G Streptococciand Staphylococci aureus. Also prepared is a second positive controlsmear comprising a suspension of only Group A Streptococci. Lastly athird smear is prepared comprising a suspension of unknown organisms tobe tested for presence of Group A Streptococci. Separately prepared isan antibody conjugate. This is made by conjugating an antibody toStreptococcus with peroxidase. The resultant conjugate is added to eachsmear which are then incubated. Thereafter, hydrogen peroxide is addedto each smear as well as an electron donor dye. The three thus treatedslide smears are observed under a microscope with the unknown smearbeing compared with the positive 'ice control to determine the presenceof Group A Streptococci. Normally, the smears prior to addition ofhydrogen peroxide and electron donor are rinsed with a buffer solutionand dried again.

In more detail, the first step in the invention involves development ofan antibody in a conventional manner by resort to a test animal such asa rabbit. A conjugate of the antibody to Group A Stroptococcus andperoxidase is then made by bridging or conjugating the two by resort toa coupling agent, such as glutaldehyde or diisocyanate. Here, typicalperoxidase enzymes which can be used are a horse radish or lactoperoxidase.

As is known each antibody has a counterpart in some antigen such as aGroup A Streptococcus. An antibody specific to an antigen will becomeattached to that antigen whenever encountered by the antibody. If anantibody that is known to be specific for a particular antigen within agroup of antibodies is isolated from the globulin portion of serum orplasma of a host animal which was stimulated to produce that antibody,then the antigen and antibody specific to that antigen will becomeattached to each oher. However, in subsequent microscopic work onecannot see the antigen-antibody organism. Thus, in the case here theperoxidase acts as a type of amplifier which will later allow theorganism to be properly observed under a microscope by resort to a dye.

A preferred method of preparing the reagent here is to form a conjugateof antibody and peroxidase, and then add the conjugate to the Group AStreptococcus. Smear slides of a swab sample and a positive and anegative control sample are prepared in the usual manner. The lowercontrol limit organisms (negative control) are prepared from aNCDC-derived panel of Group G Streptococci and Straphylococcus aureuswhich are coagulase positive, non-viable and have been reconstructedfrom a lyophilized stock. The upper control limit organisms likewise areobtained from a NCDC-derived panel of Group A Streptococci which arenon-viable and reconstituted from lyophilized stock. The upper controllimit organisms, of course, act as a positive control. The threeair-dried smears are usually rinsed in a buffer such as a tris-salinebuffer, pH 7.5 (buffer) for a few minutes and thereafter drained,usually with a filter paper. Of course, all three smears may be placedon a single slide, if desired, in separate inscribed areas.

One drop of the conjugated reagent prepared as described above is placedon each dry smear and they are incubated. In a typical situation,incubation may be carried out in a moist chamber for 20 minutes at roomtemperature. The slides are then rinsed again with buffer solution anddrained. In one specific technique the slides are immersed in a buffersolution for say about 5 minutes.

To the slides are then added a detecting dye usually an electron donordye. A typical dye useful here is 3- am1no-9-ethyl carbazole. Othersuitable dyes which may be used include 3,3'-diaminobenzidine, pchloroaniline and a mixture of u-naphthol and p-phenyldenediaminedihydrochloride. Along with the dye is added an oxidizing agent such ashyrogen peroxide. Other oxidizing agents WhlCh may be used are methylperoxide and ethyl peroxide (CH OOH; C H OOH).

In one specific technique two drops of 0.1% 3-amino- 9-ethyl carbazoledye in a buffer containing 0.3% hydrogen peroxide are added to eachsmear. Then after a suitable waiting period, say 15 minutes the slidesare again washed with tap water and each observed under a microscope.The Streptococci stains appear as a bright red stain.

In the procedure here it is believed that the peroxidase enzyme breaksdown the hydrogen peroxide. The hydrogen peroxide breakdown product inturn causes the dye to become colored, which colored material in turnbecomes attached to the Streptococci.

The following examples are illustrative of this invention.

EXAMPLE I Preparation and fixation of organisms from throat swabs Thepharyngeal mucosal swab is swirled vigorously for approximately 30seconds in 0.5 ml. saline in a Kahn or similar tube. The saline isexpressed from the swab. If desired, the same swab can be used toinoculate a blood agar plate for isolation of fi-hemolytic Streptococci.Immediately thereafter, a disposable Pasteur pipette is used to transferone drop (approximately 25 microliters) of the saline suspension ofbacteria onto the inscribed area of a microscope slide. Samples (0.01ml., 0.0001 ml.) of the same saline rinse (bacterial suspension) can betaken into a calibrated loop and spread on blood agar plates to titerthe saline wash. The test area, on the microscope slide is then allowedto air dry. The smears and fixed by immersion in absolute methanol for 5minutes and drained.

Staining technique using antibody coupled enzyme (enzyme covalentlyconjugated to antibody) Streptococcus antibody was first conjugated tohorseradish peroxidase. Two drops (approximately 50 ,ul.) of theconjugated antibody is added to each dry smear, using a Pasteur pipette.Using separate applicator sticks or toothpicks, carefully the reagent isspread over each test area. The applicator stick can be heldhorizontally to catch the meniscus of the fluid in a manner that avoidsscraping cells from the slide.

The slide is incubated at room temperature for about 20 minutes in ahumid chamber using, for example, a slide box, or an inverted petri dishwith moist paper towels fitted into or around the dish. The slide isthen rinsed with ml. of tris-saline buffer dripped from a pipette andthen washed by immersion in a staining dish of tris-saline buffer for 5minutes. The slide is dried by tilting at an angle and placing anabsorbent piece of paper (paper towels) at the edge of the inscribedarea.

Two drops (approximately 50 microliters) of the stain ing solution isthen added to each smear and allowed to react minutes. The slide isrinsed With 10 ml. distilled 'water dropped from a pipette and washed byimmersion in a staining dish filled with distilled water. The slides arethen examined for the presence of staining. At this point the slides maybe stored for several months in a microscope slide box, at roomtemperature, with no noticeable decrease in staining intensity.

The slides are examined for staining under a light microscope using a40X objective and l2.5 oculars (final magnification of 500x). Theadvantage of this procedure lies in its speed without the use of anexpensive fluorescent microscope. The relative low magnification allowsa larger area to be viewed when scanning the 1 cm. diameter circle onthe slide, thus allowing greater detection limits.

The slide areas are scanned under a light microscope at a finalmagnification of 500x. The defined detection limits are not directlyapplicable when other objectives and/ or magnification ranges are usedwith this procedure.

The positive control (Group A Streptococci) is clearly distinguishedfrom the negative control (Group G Streptococci and Staphylococcusaureas) by a bright red color. The Group A Streptococci directly fromthe throat swab stain with the same intensity and possess the samesingle cell morphology as the positive control. Primary focus must be onintensity of color and on presence and extent of chained organisms.

EXAMPLE II In a second embodiment of the invention as a variation of thetechniques described in Example I, the pharyngeal mucosal swab is placedin 1 ml. of Todd-Hewitt broth and incubated at 37 C. for 2 to 5 hours,the broth is then centrifuged 5 minutes at about 2000 r.p.m. to pack theorganisms. The supernate fluid is decanted and then the cellsresuspended in 1 ml. of saline and recentrifuged.

The saline is decanted and the tube placed in a rack for 2 to 3 minutesto allow residual saline and organisms to collect in the bottom of thetube. The organisms are mixed thoroughly in the residual saline. Using aPasteur pipette, some of the washed sediment is removed and placedwithin the inscribed area on a single slide.

The test areas are then allowed to air dry. The slide is fixed inabsolute methanol for 5 minutes and allowed to drain dry. The smears arethen processed as when the cells are eluted directly from the swab. Allcontrols and data interpretations are the same.

It is also suitable to select with an inoculating needle some organismsfrom a colon grown on a blood agar plate, emulsify the colony in salinesolution, and then transfer a drop to an inscribed slide and proceed asbefore.

What is claimed is:

1. A method of detecting the presence of Group A Streptococci in thepresence of other Streptococci and Staphyllococci organisms in anunknown sample comprising the steps of conjugating a Streptococcusantibody with a Group A Streptococcus by forming a complex of saidantibody and Group A Streptococcus through means of a peroxidase enzymecoupling agent, preparing a series of at least three slide smears; oneof such smears acting as a negative control comprising a suspension ofGroup G Streptococci and Staphylococcus aureus; a second positivecontrol smear comprising a suspension of only Group A Streptococci and athird smear comprising a suspension of unknown organisms, adding theconjugate to each smear, incubating the treated smears, adding hydrogenperoxide and an electron donor dye to the incubated treated smears, andobserving the thus treated slide smears under a microscope, said thirdunknown organism being compared with the positive control to determinethe presence of Group A Streptococci.

2. The method of claim 1 wherein the smears prior to addition ofconjugate are rinsed with a bufier solution.

3. A method of preparing a reagent useful in a method of detecting thepresence of Group A Streptococci in the presence of other Streptococciand Staphylococci organisms in an unknown sample which comprises thestep of conjugating a Streptococcus antibody with a Group AStreptococcus by forming a complex of said antibody and Group AStreptococcus through means of a peroxidase enzyme coupling agent, saidresulting conjugate being the desired reagent.

References Cited Chemical Abstracts, vol. 75, 138899e.

Chemical Abstracts, vol. 73, 220711.

Clinical and Experimental Immunology, vol. 19, No. 3, 1971, pp. 407-418.

A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner

